Transcription and translation of L1Hs elements is most abundant in human teratocarcinoma and epithelial-derived tumor cell lines. Our work is aimed at elucidating the mechanism and regulation of L1Hs transcription and translation as a basis for understanding L1Hs transposition in the human genome. We have dissected further the array of DNA segments in the L1Hs 5' untranslated region that contribute to the regulation of a downstream reporter gene. Insertion of 4 extra base pairs after residue 98 decreases gene expression more than 10-fold. The region from bp 385 to 525 enhances gene expression about 10-fold in a manner independent of either orientation or location. We have begun identifying proteins that bind to sequences in the 5' UTR as potential regulators of transcription. At least four such proteins and their binding sites have been recognized between bp 1 and 526. Two are known transcription factors: UCRBP (bp 9-21) and SP-1 (bp 426-452). The two others recognize segments 80-85 and 504-526. The difference in mobility in SDS-PAGE between the p40 polypeptides encoded by the 338 amino acid long ORF1s of two different L1Hs elements, L1.2A and cD11, is associated with four amino acid differences in the central region of p40. The L1.2A p40 has the same mobility as the endogenous p40 in teratocarcinoma cells; this finding adds to accumulating evidence indicating that L1.2A and its alleles at the LRE-1 locus on human chromosome 22 are active mobile elements. The central region of p40 includes a segment which can, in principle, form a leucine zipper in either a homo- or heterodimeric coiled-coil. Evidence that p40 in teratocarcinoma cells is in multimeric complexes has been obtained by cross-linking experiments. The endogenous p40 continues to be synthesized after differentiation of NTera2D1 cells with retinoic acid.